Spinocerebellar ataxia type 3 (SCA3), aka Machado-Joseph Disease is the most common dominantly inherited ataxia worldwide. It is the result of a CAG (glutamine) repeat expansion in the coding region of Ataxin 3, a polyglutamine (14-41 repeats)-containing protein. While investigating the mechanism of SCA3, we have found that wild type Ataxin 3 stimulates, while the mutant form abrogates the activity of polynucleotide kinase 3?- phosphatase (PNKP), an essential DNA repair protein. This resulted in the accumulation of DNA double-strand breaks in the brains of SCA3 patients and mice. Constant activation of the DNA damage-response pathway with consequent cellular apoptosis is a plausible cause of SCA3. Our recent studies have revealed that PNKP plays a critical role in DNA double strand break repair via classical non-homologous end joining (C-NHEJ). We have demonstrated that PNKP-mediated C-NHEJ repair pathway is error-free, with homologous nascent RNA providing the template to restore the missing sequence at the double strand break site in transcribed genes. DNA strand break analysis in the SCA3 mouse brain versus wild type showed significantly more strand breaks in the transcribed but, not the non-transcribed genes. Collectively, these data indicate that differential genomic region-specific strand break repair occurs in neuronal cells. Our lab found that, in addition to ATXN3, two RNA- binding/splicing proteins (NONO and SFPQ) and a key allosteric regulator of glycolysis are involved in this pathway. We are the first to show the roles of Ataxin 3 and the glycolysis-regulatory protein in classical non- homologous end joining mediated DNA double strand break repair. Notably, all these factors form a physiological complex with RNA polymerase II and other classical non-homologous end joining proteins. We also found that NONO and SFPQ significantly stimulated PNKP?s end-processing activity. We postulate that these RNA-binding proteins facilitate the formation of the RNA-DNA hybrid so that the RNA-dependent DNA polymerase can effectively use the RNA as a template to restore the missing information. However, the glycolysis inducer did not affect PNKP?s activity, but the inducer-catalyzed metabolite significantly stimulated PNKP?s activity. Furthermore, the metabolite can even restore the activity of PNKP in SCA3 patients? brain nuclear extract, suggesting the promising therapeutic potential of this natural metabolite for SCA3. Hence, this project will test the hypotheses that: restoration of the PNKP-mediated C-NHEJ repair pathway is crucial for maintaining the integrity of the transcribed genome, and thereby rescuing neuronal cells from the deleterious effects of mutant ATXN3. Understanding the mechanistic basis of error-free double strand break repair of the transcribed genes in neuronal cells via classical non-homologous end joining pathway, and the modulatory effect of a natural metabolite in such a pathway, will significantly advance our knowledge and should accelerate the development of new treatment modalities for SCA3 and other polyQ diseases.